Codon-optimized recombinant plasmid, method of stimulating peripheral nerve regeneration, and method of treating nerve damage in humans

ABSTRACT

Provided is a method for treating a peripheral nervous system damage or injury, or for regenerating peripheral nervous system tissue that involves administering to a subject in need thereof a vector that comprises polynucleotide sequences that encode vascular endothelia growth factor (VEGF) and fibroblast growth factor (FGF2) and further a polynucleotide that encodes resistance to kanamycin. A gene-therapeutic structure coding vascular endothelial growth factor (VEGF) and (FGF-2) is also provided. The gene-therapeutic structure can be administered directly to a damaged nerve and paraneural tissues both in intraoperative and post-operative period to stimulate peripheral nerve regeneration. The structure and method significantly advance existing methods for reconstructive treatment for damaged peripheral nerves.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application PCT/RU2015/000545,filed Aug. 27, 2015, which claims priority to application RU2014137218,filed Oct. 16, 2014, both of which are incorporated herein by referencein their entireties.

BACKGROUND OF THE INVENTION

Field of Invention

The present invention is in the field of medicine and more specificallyin the fields of neurosurgery, traumatology and maxillofacial surgery asapplied to treatment of peripheral nerve injuries. These injuries areeffectively treated with engineered recombinant nucleic acids. Oneexample of such an engineered recombinant nucleic acid is a plasmid thatencodes and expresses vascular endothelial growth factor (VEGF) andfibroblast growth factor (FGF2) when contacted with or transformed intoa tissue.

Discussion of the Background

About 3-10% of the population sustains peripheral nervous systeminjuries [1-3]. Peripheral nerve injuries are a common cause ofoccupational disability and such injuries not only incapacitate numerousworkers or working age individuals, but reduce the quality of life.Rehabilitation of a peripheral nerve injury can require a prolongedperiod of treatment including periods of a year or longer. Photographsof peripheral nervous system injuries and their symptoms are shown byFIGS. 2-5.

Existing methods for treating peripheral nervous system injuries dependon the extent and nature of the injury in a particular individualincluding a mechanism of the injury, extent of the peripheral nervoussystem defect, distance from the location of the injury of theperipheral nerve to the innervated area, and the time elapsed betweenthe injury and surgical intervention.

One type of the reconstructive treatment involves reconnection of theincised nerve ends by means of the end-to-end anastomosis. Peripheralnerve injuries are often accompanied by the formation of prolongeddefects, thereby rendering this approach inapplicable. In such cases,autologous nerve grafting is the most appropriate option for repairingprolonged nerve defects. A nerve that is less functionally significantcan be used as an autologous graft. Another treatment involvesreplacement of a peripheral nervous system tissue defect with variousstructures that create conditions for peripheral nerve regeneration,such as a tubular structure that is designed to replace an extendedtissue defect and foster peripheral nerve regeneration. However, despitethe advances in reconstructive techniques to restore the peripheralnerve integrity only a partial recovery of the function of an innervatedextremity usually occurs even under the most favorable conditions.

These limitations of conventional modes of treating peripheral nervoussystem injuries necessitate a further search for new methods thatenhance results of standard reconstructive treatment, reduce morbidity,disability and generally improve quality of patient's life.

One concept under study involves use of growth factors to induceregeneration of a peripheral nerve. This concept has resulted from theaccumulation of knowledge about the roles various growth factors play inthe natural process of peripheral nerve health, growth, and regeneration[4].

Vascular endothelial growth factor (VEGF) is one of the well-studiedgrowth factors that affect recovery of peripheral nerves. VEGF is one ofthe main regulators of angiogenesis and vasculogenesis. It is adisulfide-bound dimeric glycoprotein having an average molecular weightof 34-42 kDa. VEGF-A is a specific mitogen for endothelial cells (ECs)and induces their proliferation, activation, differentiation andformation of EC capillary tubules. These capillary tubules are furtherremodeled into mature blood vessels. VEGF also induces expression ofantiapoptotic proteins and increases survival of ECs. Serious defectsand improper development of the cardiovascular system occurs in animalswhere genes encoding VEGF have been deleted. These defects may be fatal.

A human VEGF is encoded by a gene located on the chromosomal locus6p21.3. The coding region comprises about 14,000 bps. VEGF has severalisoforms including VEGF 121, VEGF 145, VEGF 148, VEGF 165, VEGF 183,VEGF 189, and VEGF 206. These isoforms result from the alternativesplicing of VEGF mRNA which consists of 8 exons. Different isoforms ofVEGF have biochemical differences in the ability to bind heparin- andheparan-sulphate which permits them to traffic to differentextracellular locations. Differences in biochemical properties orextracellular trafficking of human VEGF-A isoforms are attributable tothe alternative splicing of exons 6 and 7, because all transcripts ofthe human VEGF-A gene contain exons 1-5 and 8.

VEGF had long been considered only as an inductor of angiogenesis and asa potential therapeutic agent for treatment of different disordersaccompanied by tissue ischemia. However, new data on VEGF'sneuroprotective properties for neurons of both the peripheral andcentral nervous systems have been obtained [5, 6]. VEGF stimulatesproliferation of Schwann cells, astrocytes, microglia, and corticalneurons [7-10]. A significant increase of expression of VEGF and Flt-1(VEGF type II receptor) in the lumbar spine in response to an injury wasshown in a rat sciatic nerve crush injury model [11]. The axonalsprouting that manifests as the increased axon number in the conduit pera unit of the cross section area was observed when VEGF was used as apart of the matrigel filling in the conduit [12].

The use of VEGF-loaded poly-lactic acid microspheres in an autologousvein graft in a model of trauma with an extensive defect of fibular andtibial nerves was found to improve the nerve functional index and toincrease the number of myelinated fibers in the graft [13].

VEGF has been shown to induce Schwann cell division and migration in agraft towards distal parts that correlates with the increased number ofcapillaries and myelinated fibers [14].

Introduction of VEGF in combination with a Brain-derived neurotrophicfactor (BDNF) into cavernosal bodies in a rat cavernous nerve injurymodel resulted in the recovery of the lost innervation and erectilefunction [15].

FGF is another growth factor that induces neurogenesis. FGF inducesSchwann cell proliferation and migration in a peripheral nerve injury[16].

In experiments using animal models, it was shown that blocking receptorsfor FGF, Fgfr1 and Fgfr2, caused neuropathy of non-myelinating sensoryfibers and significant impairment of the thermal pain sensitivity [17].

The use of bone marrow-derived stem cells in a peripheral nerve injurymodel resulted in increased FGF expression that induced migration andproliferation of Schwann cells [18].

In a thoracic spinal cord injury model, the use of FGF in a sciaticnerve graft promoted the improvement of the upper extremity motorfunction [19].

Therapeutic applications of growth factors, such as VEFG and FGF, wereknown to have a number of limitations. After the administration into theinjury site the growth factors undergo rapid degradation and, therefore,their constant concentration cannot be maintained to achieve the desiredtherapeutic effect [20].

Gene therapy using vectors that express growth factors like VEGF hadpreviously been performed. There are two main trends in gene therapy:(i) use of viral vectors and (ii) use of non-viral vectors. Thesedifferent trends generally operate through different mechanisms of genetransfer. The use of viral vectors in the clinical setting, despitetheir high transfection activity, is limited due to the risk ofinsertional mutagenesis and potential induction of the inflammatoryresponse and toxicity.

A safer method of gene transfer is based on the use of plasmid DNA. In amodel of musculocutaneous nerve repair with end-to-end and end-to-sideanastomosis, intraoperative administration of a DNA plasmid comprising avegf gene into a distal region resulted in the significantly increasednumber of myelinated fibers per a unit of the cross-section area of theregion distal to the anastomosis site that correlated with a significantincrease of the VEGF concentration in Schwann cells [21].

A gene-therapeutic construction could be injected paraneurally. In asciatic nerve injury model, plVEGF was administrated intramuscularly andwas combined with a hyaluronic acid film sheath which covered theanastomosis site in order to reduce severity of the scarring. The drugintramuscular injection was accompanied by a significant increase of themuscular response amplitude and the increased number of myelinatedfibers distal to the anastomosis site against their use as monotherapy[22].

The study performed by Wang F. et al. demonstrated a plVEGFdose-dependent effect when the gene therapeutic construction was givenintraneurally after the end-to-end suturing of sciatic nerve stumps. Theuse of a higher dosage resulted in the most pronounced increase ofneurophysiological parameters and a lesser decrease of the calf muscleweight index [23].

Synergism in action of some factors has been uncovered. For example,combined use of a VEGF gene-coding plasmid and a plasmid encoding theC-CSF gene in a sciatic nerve injury model demonstrated a morepronounced increase in the number of myelinated fibers and capillariesin the region distal to the end-to-end anastomosis, maintenance of moreneurons in the spinal ganglia as well as the early recovery of the motorfunction [24]. However, only a part of the cells is transfected withplasmid DNA when using gene therapeutic agents in vivo. Consequently,the probability that a cell is transfected simultaneously with twodifferent gene therapeutic constructions is reduced. The efficacy of acombination of genetic sequences of two growth factors having asynergistic action in one plasmid has been demonstrated in an animalmodel of the spinal cord contusion injury.

During this experiment it had been shown that when 40 μg of a VEGF andFGF2 gene containing plasmid were directly injected into the spinalcord, there was a significant increase of the capillary number in thesections made at 1.5 cm from the trauma core. Based on the behavior testdata, the recovery of the motor function significantly improved ascompared to the control group of animals that were not given the plasmidcontaining VEGF and FGF2 genes. Based on the results obtained in thisexperiment, it had been concluded that application of the doublecassette plasmid improves spinal cord vascularization and reduces thearea of destruction of the spinal gray and white matter [25].

The use of gene therapeutic constructions comprising VEGF and the basicfibroblast growth factor to improve sciatic nerve recovery has beendescribed in [26]. Patent RU 2459630 C1 “Stimulation Technique forNeuroregeneration with Genetic Constructions” describes a method of thepost-traumatic regeneration of the rat spinal cord when injecting adouble-cassette plasmid pBud-VEGF-FGF2.

Spinal cord and peripheral nerves exhibit significant differences in theregenerative potential. Consequently, the results described above havegiven no indication as to whether the treatment of a peripheral nerveinjury with a plasmid expressing both VEGF and FGF2 could be effectivein repairing peripheral nerve injuries. This is due to the fact that themechanism of a contusion injury significantly differs by pathogenesisand a degree of severity from a trauma accompanied with neurotmesiswhich is more specific for peripheral nerves and prevails in the totalstructure of their injuries. Moreover, as mentioned above, the type orextent of regenerative or recuperative effects of applying particulargrowth factors, or a combination of growth factors, to damagedperipheral nervous system tissue are substantially unexplored.

Keeping in mind these problems with protein-based therapies anduncertainties regarding responsiveness of peripheral nerve injuries to anucleic-acid based therapy, the inventors have developed nucleic-acidbased vectors that express growth factors such as VEGF and FGF2 andinitiated studies to determine whether incorporation of these growthfactors into a complex therapy of a peripheral nerve repair could beeffective. As shown herein, a better, more reliable, and more effectivetreatment of peripheral nerve injuries is possible using a nucleicacid-based therapy.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodfor treating a peripheral nervous system damage or injury, or forregenerating peripheral nervous system tissue, comprising administeringto a subject in need thereof a vector that comprises polynucleotidesequences that encode vascular endothelia growth factor (VEGF) andfibroblast growth factor (FGF2).

In one embodiment, the vector comprises FGF2 encoding nucleotides atpositions 699-1166 and VEGF165 encoding nucleotides at positions3723-4298 of SEQ ID NO: 1 and resistance to kanamycin nucleotides atpositions 1469-2511 of SEQ ID NO: 1. In another embodiment, the vectoris pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1).

Another object of the present invention is to provide a vectorcomprising polynucleotide sequences that encode vascular endotheliagrowth factor (VEGF), fibroblast growth factor (FGF2), and resistance tokanamycin. In one embodiment, the vector has SEQ ID NO: 1 and comprisesFGF2 encoding nucleotides at positions 699-1166, VEGF165 encodingnucleotides at positions 3723-4298 of SEQ ID NO: 1, and resistance tokanamycin nucleotides at positions 1469-2511 of SEQ ID NO: 1.

Another object of the present invention is to provide a cell that hasbeen transformed with the vector.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 provides the repeated approach. The entire sciatic nerve with anautologous graft is visualized. Results after injection of plasmidpBud(Kan)-VEGF-FGF2.

FIG. 2 shows a view of the upper extremity prior to surgery.Post-traumatic and post-operative indented irregular scars are seen onthe anterior and posterolateral surfaces of the lower, middle, and upperthird of the right upper arm.

FIG. 3 shows lack of active movements in the middle phalanges of fingers2-5.

FIG. 4 shows the impaired prehension function by all fingers.

FIG. 5 shows high-grade atrophy of the hand muscle within a zoneinnervated by the median and ulnar nerves and the ability to opposefinger 1 to finger 2 only.

FIG. 6 shows injection of the recombinant plasmid pBud (Kan)-VEGF-FGF2into the repaired nerve into the suture zone and also proximally anddistally over the length of 10 cm.

FIG. 7 shows application of fibrin glue to prevent leakage of therecombinant plasmid.

FIG. 8 shows atrophy of hand and forearm muscles. Nail changes:hypoplastic. Secretory function (sweating): decreased. Figuredemonstrates post-surgical improvement in patient's condition.

FIG. 9 shows a hook grasp (a purse handle). Figure demonstratespost-surgical improvement in patient's condition.

FIG. 10 shows a first grasp. Figure demonstrates post-surgicalimprovement in patient's condition.

FIG. 11 shows tip prehension (finger Figure demonstrates post-surgicalimprovement in patient's condition.

FIG. 12 shows tip prehension (finger Figure demonstrates post-surgicalimprovement in patient's condition.

FIG. 13 shows tip prehension (finger I-IV). Figure demonstratespost-surgical improvement in patient's condition.

FIG. 14 shows a diagram of electromyography results for the thenarmuscle group. Based on the electromyography results the thenar muscleresponse amplitude had increased over the year from 0 mV to 5 mV andalmost achieved the value of the contralateral extremity.

FIG. 15 shows electromyography findings for the hypothenar muscle group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is used in medicine, preferably in neurosurgery,traumatology and maxillofacial surgery, and in treatment of peripheralnerve injuries.

As used herein the words “a”, “an” and the like generally carry ameaning of “one or more”, unless stated otherwise.

A goal of the inventors' research has been to create, based on theirexperience in the development of gene therapeutic agents, an effectiveproduct for treating patients with peripheral nerve injuries. For thispurpose, the inventors have developed various gene therapeuticconstructions that differ from each other by the number of encodedtransgenes and the transgenes, as well as by the nucleotide sequences ofthe same transgenes.

In one embodiment, an object of the present invention is to provide animproved or enhanced method for reconstructive treatment involvingdelivery of a therapeutic polynucleotide construct into or in thevicinity of a damaged peripheral nervous system tissue. An example ofthis embodiment is the delivery of genetic sequences encoding VEGF andFGF-2 into such tissue using the recombinant plasmidpBud(Kan)-VEGF-FGF2.

An object of the present invention is to provide a method for treating aperipheral nervous system damage or injury, or for regeneratingperipheral nervous system tissue, comprising administering to a subjectin need thereof a vector that comprises polynucleotide sequences thatencode vascular endothelia growth factor (VEGF) and fibroblast growthfactor (FGF2).

A range of the injected plasmid could be from 200 to 500 μg per nerve in2.5 ml of a physiologic saline solution. The ranges include all valuesand subranges therebetween, including 250, 300, 350, 400, and 450 μg pernerve in 2.5 ml of a physiologic saline solution and any amount inbetween.

The vector could be administered in vivo. In another embodiment, thevector is administered to a site of the peripheral nervous system damageor injury or to a tissue to be regenerated. In a different embodiment,the vector is administered to a site of the peripheral nervous systemdamage or injury at a site proximal or distal to the peripheral nervoussystem damage, or at sites proximal and distal to said damage. Thevector could be administered intra-, peri- and/or paraneurally.

In yet another embodiment, the vector is contacted with a neuron or aSchwann cell, astrocyte, microglia and/or neuron.

In one embodiment, the subject has neurotmesis. In another embodiment,the subject has a diastatic peripheral nerve damage. In a differentembodiment, the subject has peripheral nerve damage other thanneurotmesis or diastatic peripheral nerve damage. The subject could behuman or animal.

In one embodiment, the vector comprises a polynucleotide sequence thatencodes resistance to kanamycin.

In another embodiment, the vector comprises FGF2 encoding nucleotides atpositions 699-1166 and VEGF165 encoding nucleotides at positions3723-4298 of SEQ ID NO: 1. The vector further could comprise resistanceto kanamycin nucleotides at positions 1469-2511 of SEQ ID NO: 1. In yetanother embodiment, the vector is pBud(Kan)-VEGF-FGF2 that has SEQ IDNO: 1.

Another object of the present invention is to provide a vectorcomprising polynucleotide sequences that encode vascular endotheliagrowth factor (VEGF), fibroblast growth factor (FGF2), and resistance tokanamycin. In one embodiment, the vector comprises FGF2 encodingnucleotides at positions 699-1166 and VEGF165 encoding nucleotides atpositions 3723-4298 of SEQ ID NO: 1. In another embodiment, the vectorhas SEQ ID NO: 1.

A different object of the present invention is to provide a cell thathas been transformed with the vector.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Examples Comparative Example 1. Treatment of Diastatic Peripheral NerveInjury with VEGF/FGF2 Gene Therapy

An animal model of a diastatic peripheral nerve injury was used toevaluate effects of gene therapy with the plasmid vector encoding bothVEGF and FGF2 described by Masgutov [26].

Test animals (rats), were divided into three groups: (i) intact group,(ii) a test group where a gene therapeutic construction wasadministered, and (iii) a control group where a phosphate-bufferedsaline (PBS) solution was injected instead of the gene therapeuticconstruction.

In test group (ii) a total dose of 45 μg of a gene therapeuticconstruction was directly injected equally into distal and proximal endsof an autologous nerve graft. In control group (iii) aphosphate-buffered saline (PBS) solution was injected into theselocations instead of the gene therapeutic construction.

The evaluation criteria of the regeneration dynamics of the peripheralnerve included neurophysiological parameters such as the nerveconduction velocity and the muscle response amplitude as well as thehistological examination findings such as the number of myelinatedfibers and the capillary network density.

On day 56 following the injection of the plasmid construction, theneurophysiological parameters in the test group (ii) were superior tothose in the control group (iii); however, they were significantlyinferior to those in the intact animals of group (i).

A histological examination revealed that myelinated fiber numbers perunit of the cross-section area of the graft were significantly higher inthe experimental group (ii) compared to the control group (iii).However, no effective recovery of the extremity function was observed.These experiment results show that the use of plasmid-basedconstructions containing genetic sequences of growth factors provides astimulating effect on the regeneration of peripheral nerves.

Example 1. Construction and Evaluation of Vector Encoding VEGF and FGF2in Animal Model

The inventors have sought to determine whether the effect observed inComparative Example 1 was attributable to the construction of the usedplasmid. The inventors have engineered a new plasmid encoding VEGF andFGF2 which replaced the tag sequences in the prior vector with a geneencoding kanamycin resistance. Among other constructs, plasmidpBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) was constructed. This plasmid hasbeen engineered to include a sequence encoding resistance to kanamycinat nucleotides 1469-2511 of SEQ ID NO: 1; cDNA of a gene encoding FGF2at nucleotides 699-1166 in SEQ ID NO: 1; cDNA of the gene encodingVEGF165 at nucleotides 3723-4298 in SEQ ID NO: 1; and the Kozak sequenceat nucleotides 695-698 and 3719-3722.

The rat animal model of a peripheral nerve injury substantially asdescribed in Comparative Example 1 was used to evaluate the effect ofadministering the new plasmid constructs, including plasmidpBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1). Gene therapeutic constructions wereadministrated intraneurally immediately after the peripheral nervesuturing. The results were evaluated after 60 days following thesurgical intervention and therapeutic constructs administration. Of allthe plasmid DNAs that were used, the best results were obtained for theplasmid pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) containing genetic sequencesof FGF2 and VEGF. The results for the plasmid pBud(Kan)-VEGF-FGF2 aredepicted by FIG. 1. Based on these favorable non-clinical results, theinventors evaluated whether peripheral nerve regeneration could beattained in the clinical setting using the gene therapeutic constructionpBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1), as described below.

Example 2. Clinical Evaluation of Regenerative Effects ofpBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1)

Patient B., born in 1985, was admitted to the trauma center of theRepublic Clinical Hospital of MoH of the Republic of Tatarstan on Apr.4, 2011, with the diagnosis of sequelae of the median and ulnar nerveinjury in the middle third of the right upper arm as shown by FIG. 2.From the patient's history, it was known that in 2009 the patient had aglass cut of the middle third of the upper arm, with the median andulnar nerves damaged.

The median and ulnar nerves were sutured end-to-end immediately afterthe injury. However, both motor and sensitivity functions were completedabsent in the immediate post-operation period. A course ofrehabilitation therapy had produced no visible results.

After 7 months, in 2010, neurolysis of the median and ulnar nerves wasperformed due to the lack of positive changes in the motor andsensitivity functional recovery. Slight changes in nerve regenerationwere observed in the post-operative follow-up, namely, complete lack ofsensitivity, at the same time, the motor function appeared which wascharacterized by the mild bending of the injured hand and fingers. Itwas decided to perform a surgical treatment.

Prior to surgery, on Apr. 21, 2011, the patient had an examination withthe following results:

Trophic Disturbances

-   -   a) skin status: normal color, decreased fingers' temperature,        increased feeling of chillness;    -   b) atrophy of the hand and forearm muscles, as compared to the        normal arm: more than 2 cm as shown by FIGS. 2 and 3.    -   c) nail changes: hypoplastic; and    -   d) secretory function (sweating): decreased.

Sensitivity testing in the patient in the autonomous zone of innervationby the nerve:

No. Kinds of Sensitivity Brief Description 1. Pain Absent 2. TemperatureAbsent 3. Tactile Absent 4. Discriminative Absent 5. Sense oftwo-dimension space Absent 6. Stereognosis Absent 7. Sense of pressureAbsent 8. Sense of weight Absent

Degree Sensitivity recovery S0 Lack of sensitivity within the nerveautonomous zone

Motor function testing

Degree Motor function recovery M2 Distinct contractions withoutmovements in joints

Hand prehension patterns: the hand is unable to perform any type ofprehension (FIG. 3-4).

Diagnosis: the injury of the median and ulnar nerves in the middle thirdof the forearm sustained 2 years ago. The status post suturing andneurolysis of the median and ulnar nerves are shown in FIG. 5.

A surgery was performed on Apr. 26, 2011, including neurolysis of themedian and ulnar nerves with the intraneural administration of plasmidpBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) containing the vegf and fgf-2 genes.

The surgery was conducted under the nerve block anaesthesia. Followingtriple treatment of the surgical field, an arcuate incision was made onthe inner surface of the right upper arm. The median and ulnar nerveswere isolated with technical difficulties. The suture lines had beenfound. There were no neuroma signs observed; however, the nerves wereinvolved in a scar-forming process and adhered to the surroundingtissue.

The plasmid pBud(Kan)-VEGF-FGF2 was injected with an insulin needle, 250μg per nerve in 2.5 ml of a physiologic saline solution. The injectionwas administered into the suture zone and also proximally and distallyover the length of 10 cm (FIG. 6). After that 2 ml of the two-componentfibrin glue TISSUCOL® was applied to the isolated nerves (FIG. 7).

The post-surgical case included hemostasis, wound suturing, placement ofa rubber tube drainage, and application of an antiseptic dressing and aplaster cast. A re-examination was performed one month after thesurgery.

The results of the physical examination dated on May 25, 2011, arepresented below:

Trophic disturbances

-   -   a) skin status: normal color;    -   b) atrophy of the injured hand and forearm muscles, compared to        the normal arm: more than 2 cm (FIG. 8);    -   c) nail changes: hypoplastic; and    -   d) secretory function (sweating): decreased.

Sensitivity testing in the patient in the autonomous zone of innervationby the nerve:

No. Kinds of Sensitivity Brief Description 1. Pain Absent 2. TemperatureHot - absent Cold - distal phalanges of fingers 1, 2 3. Tactile Fingers1, 2 - distal phalange 4. Discriminative Absent 5. Sense oftwo-dimension space Absent (Moberg pickup test) 6. Sense of pressurePresent 7. Sense of weight present

Degree Sensitivity recovery S1 Recovery of deep pain sensitivity withinthe nerve autonomous zone

Motor function testing

Degree Motor function recovery M2 Distinct contractions withoutmovements in joints

Hand prehension patterns: the hand is unable to perform any type ofprehension.

A regular examination was performed in 6 months after the surgery. Theresults of the physical examination dated on Nov. 15, 2012, arepresented below:

Trophic disturbances

-   -   a) skin status: of normal color;    -   b) atrophy of the hand and forearm muscles, compared to the        normal arm: moderate (1-2 cm) and severe (more than 2 cm);    -   c) nail changes: within normal limits; and    -   d) secretory function (sweating): normal.

Sensitivity testing in the patient in the autonomous zone of innervationby the nerve:

No. Kinds of Sensitivity Brief Description 1. Pain Present, includingdistal phalanges of all fingers 2. Temperature Hot - distal phalanges offinger 1, middle phalanges of fingers 3, 4 Cold - distal phalanges offingers 1, 3; distal phalanges of fingers 2, 4, 5 3. Tactile Fingers 1,3 - distal phalange, middle phalange 2, 4, 5 4. Discriminative finger1-10 mm finger 2-30 mm finger 3-20 mm finger 4-30 mm finger 5-30 mm 5.Sense of two-dimension space Identifies large objects (a box of (Mobergpickup test) cigarettes, glue, tube for blood collection), a pencil,glue tube 6. Sense of pressure Present 7. Sense of weight present

Degree Sensitivity recovery S3 Recovery of surface pain and tactilesensitivity within the entire autonomous zone with complete hyperpathiadisappearance

Motor function testing

Degree Motor function recovery M3 Mild movements in joints (usefulrecovery)

Hand prehension patterns:

1) cylindrical grasp—YES;

2) spherical grasp—YES;

3) hook grasp (a bag handle)—YES;

4) first grasp—YES;

5) tip prehension

-   -   a) terminal opposition—YES;    -   b) subterminal opposition—NO;

6) lateral prehension

-   -   a) pinch grip—NO;    -   b) scissor grip—“cigarette”—NO.

A year after the surgery, the patient had a regular examination. Theresults of the physical examination dated on Apr. 20, 2012, arepresented below:

Trophic disturbances

-   -   a) skin status: of normal color;    -   b) atrophy of the injured hand and forearm muscles, compared to        the normal arm: moderate (1-2 cm);    -   c) nail changes: within normal limits; and    -   d) secretory function: within normal limits.

Sensitivity testing in the patient in the autonomous zone of innervationby the nerve:

No. Kinds of Sensitivity Brief Description 1. Pain Present, includingdistal phalanges of all fingers 2. Temperature Hot - distal phalanges offingers 1 and 3, middle phalanges of fingers 4, 5 Cold - distalphalanges of fingers 1, 3; distal phalanges of fingers 1, 2, 3, 4, 5 3.Tactile Fingers 1, 2, 3, 4, 5 - distal phalange 4. Discriminative finger1-5 mm finger 2-5 mm finger 3-10 mm finger 4-5 mm finger 5-10 mm 5.Sense of two-dimension space Identifies large objects (a box of (Mobergpickup test) cigarettes, glue, tube for blood collection), as well assmall objects (rubber, button, coin, clip) 6. Sense of pressure Present7. Sense of weight present

Degree Sensitivity recovery S3+ Recovery of surface pain and tactilesensitivity within the entire autonomous zone with complete hyperpathiadisappearance, but with some recovery of two-point discrimination withinthe autonomous zone (from 12 to 15 mm)

Motor function testing

Degree Motor function recovery M4 Movements with overcoming someresistance

Hand prehension patterns:

1) spherical grasp—YES;

2) spherical grasp—YES;

3) hook grasp—YES (FIG. 9);

4) first grasp—YES (FIG. 10);

5) tip prehension: (FIG. 11-13)

-   -   a) terminal opposition—YES;    -   b) subterminal opposition—YES;

6) lateral prehension

-   -   a) pinch grip—YES;    -   δ) scissor grip—YES.

These clinical results show that the extremity function wassignificantly improved one year after the intraneural administration ofthe gene-therapeutic construction containing a plasmid expressing VEFGand FGF2. The improved functional state of the extremity was manifestedas the decreased severity of the trophic disturbances, as thedevelopment of various sensitivities within the area of innervation ofthe median and ulnar nerves, and as a significant improvement of themotor function. Based on the electromyography results the thenar muscleresponse amplitude had increased over the year from 0 mV to 5 mV andalmost achieved the value of the contralateral extremity (FIGS. 14 and15).

An animal model and clinical results show that a plasmid that expressestwo growth factors, VEGF and FGF2, provides a more effective inductionof the peripheral nerve regeneration that prior plasmid constructs.

The efficacy of usinf a gene therapeutic construction to improve resultsof surgical treatment of peripheral nerve injuries has been determinedand demonstrated by the present inventors in the above describedexperiments and clinical observations. While not being bound to anyparticular mechanism, the inventors believe that the achieved clinicaleffects when using the plasmid pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) werelikely obtained due to the combination of these two growth factors.However, a full understanding of the influence of genetic constructsrequires further studies.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.Further, the materials, methods, and examples are illustrative only andare not intended to be limiting, unless otherwise specified.

Numerous modification and variations on the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

REFERENCES

-   1. Hudso, A. R. Timing of peripheral nerve repair: important local    and neuropathological factors/A. R. Hudson//Clinical    Neurosurgery.—1977.—Vol. 24.—C. 391-405.-   2. Deitch, E. A. Experience with 112 shotgun wounds of the    extremities/E. A. Deitch, W. R. Grimes//J Trauma.—1984.—Vol. 24.—P.    600-603.-   3. Analysis of upper and lower extremity peripheral nerve injuries    in a population of patients with multiple injuries/C. A. Munro//J.    Trauma.—1998.—Vol. 45.—P. 116-122.-   4. Terenghi, G. Peripheral nerve regeneration and neurotrophic    factors/G. Terenghi//J. Anat. 1999.—Vol. 194.—P.-   5. Vascular endothelial growth factor has neurotrophic activity and    stimulates axonal outgrowth, enhancing cell survival and Schwann    cell proliferation in the peripheral nervous system/M. Sondell, M.    Kanje. G. Lundborg//J Neurosci.—1999.—Vol. 19, NO 14—P. 5731-40.-   6. VEGF-A165b is an endogenous neuroprotective splice isoform of    vascular endothelial growth factor A in vivo and in vitro/N.    Beazley-Long [et al.]//J Pathol.—2013.—Vol. 183, No 3—P. 918-29.-   7. Sondell, M. Vascular endothelial growth factor stimulates Schwann    cell invasion and neovascularisation of acellular nerve grafts/M.    Sondell, G. Lundborg, M. Kanje//Brain Res.—1999.—Vol. 846—P.    219-228.-   8. Vascular, glial and neuronal effects of vascular endothelial    growth factor in mesencephalic expiants cultures/W. F. Silverman [et    al.]//Neuroscience.—1999.—Vol. 90—P. 1529-1541.-   9. Forstreuter, F. Vascular endothelial growth factor induces    chemotaxis and proliferation of microglial cells/F. Forstreuter, R.    Lucius, R. Mentlein//J. Neuroimmunol.—2002.—Vol. 132—P. 93-98.-   10. Zhu, Y. Vascular endothelial growth factor promotes    proliferation of cortical neuron precursors by regulating E2F    expression/Y. Zhu [et al.]//J FASEB.—2003. Vol. 17—P. 186-193.-   11. Induction of VEGF and its Flt-1 receptor after sciatic nerve    crush injury/R. R. Islamov [et al.]//Neuroreport.—2004. Vol. 15, No    13—P. 2117-21.-   12. Effects of vascular endothelial growth factor on nerve    regeneration in acellular nerve grafts/J. M. Rovak [et al.]//J    Reconstr Microsurg.—2004. Vol. 20, No 1—P. 53-58.-   13. Vascular endothelial growth factor-loaded poly    (lactic-co-glycolic acid) microspheres-induced lateral axonal    sprouting into the vein graft bridging two healthy nerves: nerve    graft prefabrication using controlled release system/H. Karagoz [et    al.]//J Microsurgery.—2012. Vol. 32, No 8—P. 635-41.-   14. Sondell, M. Vascular endothelial growth factor stimulates    Schwann cell invasion and neovascularization of acellular nerve    grafts//M. Sondell, G. Lundborg, M. Kanje//Brain Res.—1999. Vol.    846, No 2—P. 219-28.-   15. The effect of vascular endothelial growth factor and    brain-derived neurotrophic factor on cavernosal nerve regeneration    in a nerve-crush rat model/PS. Hsieh [et al.]//BJU Int.—2003. Vol.    92, No 4—P. 470-5.-   16. Grothe, C. Physiological function and putative therapeutic    impact of the FGF-2 system in peripheral nerve regeneration-lessons    from in vivo studies in mice and rats/K. Haastert, J. Jungnickel, C.    Grothe//Brain Res Rev.—2006. Vol. 51—P. 293-299.-   17. Furushol, M. Disruption of Fibroblast growth factor receptor    signaling in non-myelinating Schwann cells causes sensory axonal    neuropathy and impairment of thermal pain sensitivity/M. Furushol    [et al.]//J Neurosci.—2009. Vol. 29, No 6—P. 1608-1614.-   18. Tulio, V. R. Bone marrow-derived fibroblast growth factor-2    induces glial cell proliferation in the regenerating peripheral    nervous system/V. R. Tulio [et al.]//Molecular    Neurodegeneration.—2012. Vol. 7, No 34—P. 1-17.-   19. Sciatic nerve grafting and inoculation of FGF-2 promotes    improvement of motor behavior and fiber regrowth in rats with spinal    cord transaction/F. P. Guzen, [et al.]//Restorative Neurology and    Neuroscience.—2012. Vol. 30—P. 265-275.-   20. Zeng, W. Ionically cross-linked chitosan microspheres for    controlled release of bioactive nerve growth factor/W. Zeng [et    al.]//Int J Pharm.—2011. Vol. 421—P. 283-290.-   21. Enhancement of musculocutaneous nerve reinnervation after    vascular endothelial growthfactor (VEGF) gene therapy/P. Haninec [et    al.]//BMC Neuroscience.—2012. Vol. 13, No 57—P.-   22. Effect of VEGF gene therapy and hyaluronic acid film sheath on    peripheral nerve regeneration/F. Zor [et al.]//—2014. Vol. 34, No    3—P. 209-16.-   23. Favorable effect of local VEGF gene injection on axonal    regeneration in the rat sciatic nerve/C. Fu [et al.]//J Huazhong    University Scince Technology.—2007. Vol. 2—P. 186-9.-   24. Double gene therapy with granulocyte colony-stimulating factor    and vascular endothelial growth factor acts synergistically to    improve nerve regeneration and functional outcome after sciatic    nerve injury in mice/F. Pereira Lopes [et al.]//Neuroscience. —2013.    Vol. 230—P. 184-97.-   25. Pat. 2459630 RF, IPC A61K 48/00, A61P 25/28, C12N 15/79, C1.    Stimulation Technique for Neuroregeneration with Genetic    Constructions/Yu. A. Chelyshev; Federal State Educational    Institution “Kazan Federal University”.—No 2011116853/10;    27.04.2011; published 27 Oct. 2009, Bull. No 30.—11 p. [in Russian]-   26. Stimulation of post-traumatic regeneration of a rat sciatic    nerve with a plasmid expressing the vascular endothelial growth    factor and the basic fibroblast growth factor./R. F. Masgutov [et    al]//Cell Technologies and Tissue Engineering.—2011.—6(3): 67-70.    [in Russian].

1. A method for treating a peripheral nervous system damage or injury,or for regenerating peripheral nervous system tissue, the methodcomprising administering to a subject in need thereof a vector thatcomprises polynucleotide sequences that encode vascular endotheliagrowth factor (VEGF) and fibroblast growth factor (FGF2).
 2. The methodof claim 1, for treating a peripheral nervous system damage or injury.3. The method of claim 1 that is directed to regenerating peripheralnervous system tissue.
 4. The method of claim 1, wherein the vector isadministered in vivo.
 5. The method of claim 1, wherein the vector isadministered to a site of the peripheral nervous system damage or injuryor to a tissue to be regenerated.
 6. The method of claim 1, wherein thevector is administered to a site of the peripheral nervous system damageor injury at a site proximal or distal to the peripheral nervous systemdamage, or at sites proximal and distal to said damage.
 7. The method ofclaim 1, comprising administering the vector intra-, peri- and/orparaneurally.
 8. The method of claim 1, comprising contacting the vectorwith a neuron or a Schwann cell, astrocyte, microglia and/or neuron. 9.The method of claim 1, wherein the subject has neurotmesis.
 10. Themethod of claim 1, wherein the subject has a diastatic peripheral nervedamage.
 11. The method of claim 1, wherein the subject has peripheralnerve damage other than neurotmesis or diastatic peripheral nervedamage.
 12. The method of claim 1, wherein the subject is human.
 13. Themethod of claim 1, wherein the vector further comprises a polynucleotidesequence that encodes resistance to kanamycin.
 14. The method of claim1, wherein the vector comprises FGF2 encoding nucleotides at positions699-1166 and VEGF165 encoding nucleotides at positions 3723-4298 of SEQID NO:
 1. 15. The method of claim 1, wherein the vector comprisesresistance to kanamycin nucleotides at positions 1469-2511 of SEQ IDNO:
 1. 16. The method of claim 1, wherein the vector ispBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1).
 16. A vector comprisingpolynucleotide sequences that encode vascular endothelia growth factor(VEGF), fibroblast growth factor (FGF2), and resistance to kanamycin.17. The vector of claim 16 that comprises FGF2 encoding nucleotides atpositions 699-1166 and VEGF165 encoding nucleotides at positions3723-4298 of SEQ ID NO:
 1. 18. The vector of claim 16 having SEQ IDNO:
 1. 19. A cell that has been transformed with the vector of claim 16.